Development of a bifunctional CD1d-anti-HER2 scFv fusion molecule to redirect the iNKT cell-mediated immune response to the tumor site

Abstract : Invariant natural killer T lymphocytes (iNKT) are a unique subpopulation of T lymphocytes recognizing glycolipid antigens in the context of the MHC class I-like molecule CD1d. Upon activation with the high affinity ligand α-galactosylceramide (αGalCer), iNKT cells rapidly produce large amounts of the pro-inflammatory cytokine interferon gamma (IFN-γ) and potently activate cells of the innate and adaptive immune response, such as dendritic cells (DCs), NK and T cells. In this context, iNKT cells have been shown to efficiently mediate antitumor activity, and recent research has focused on the manipulation of these cells for antitumor therapies. However, a major drawback of αGalCer as a free drug is that a single injection of this ligand leads to a short-lived iNKT cell activation followed by a long-term anergy, limiting its therapeutic use. In contrast, we demonstrate here that when αGalCer is loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections lead to a sustained iNKT and NK cell activation associated with IFN-γ secretion as well as with DC maturation. Most importantly, when the αGalCer/sCD1d is fused to an anti-HER2 scFv antibody fragment, potent inhibition of experimental lung metastasis and established subcutaneous tumors is obtained when systemic treatment is started two to seven days after the injection of HER2-expressing B16 melanoma cells, whereas at this time free αGalCer has no effect. The antitumor activity of the sCD1d-anti-HER2 fusion protein is associated with HER2-specific tumor localization and accumulation of iNKT, NK and T cells at the tumor site. Importantly, active T cell immunization combined with the sCD1d-anti-HER2 treatment leads to the accumulation of antigen-specific CD8 T cells exclusively in HER2-expressing tumors, resulting in potent tumor inhibition. In conclusion, sustained activation and tumor targeting of iNKT cells by recombinant αGalCer/sCD1d molecules thus may promote a combined innate and adaptive immune response at the tumor site that may prove to be effective in cancer immunotherapy. RESUME : Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines du complexe majeur d'histocompatibilité de classe I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. En revanche, l'étude présentée ici démontre que, si l'αGalCer est chargé sur des molécules récombinantes soluble CD1d (αGalCer/sCDld), des injections répétées aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si on fusionne la molécule αGalCer/sCD1d avec un fragment single-chain (scFv) de l'anticorps anti-HER2, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. De plus, une immunisation active combinée avec le traitement sCD1d-anti-HER2 aboutit à une accumulation des lymphocytes T CD8 spécifiques de l'antigène d'immunisation, ceci exclusivement dans des tumeurs qui expriment l'antigène HER2. Cette combinaison résulte dans une activité anti-tumeur accrue. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules recombinantes αGalCer/sCDld conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer. RESUME POUR UN LARGE PUBLIC : Le cancer est une cause majeure de décès dans le monde. Sur un total de 58 millions de décès enregistrés au niveau mondial en 2005, 7,6 millions (soit 13%) étaient dus au cancer. Les principaux traitements de nombreux cancers sont la chirurgie, en association avec la radiothérapie et la chimiothérapie. Néanmoins, ces traitements nuisent aussi aux cellules normales de notre corps et parfois, ils ne suffisent pas pour éliminer définitivement une tumeur. L'immunothérapie est l'une des nouvelles approches pour la lutte contre le cancer et elle vise à exploiter la spécificité du système immunitaire qui peut distinguer des cellules normales et tumorales. Une cellule exprimant un marqueur tumoral (antigène) peut être reconnue par le système immunitaire humoral (anticorps) et/ou cellulaire, induisant une réponse spécifique contre la tumeur. L'immunothérapie peut s'appuyer alors sur la perfusion d'anticorps monoclonaux dirigés contre des antigènes tumoraux, par exemple les anticorps dirigés contre les protéines oncogéniques Her-2/neu dans le cancer du sein. Ces anticorps ont le grand avantage de spécifiquement se localiser à la tumeur et d'induire la lyse ou d'inhiber la prolifération des cellules tumorales exprimant l'antigène. Aujourd'hui, six anticorps monoclonaux non-conjugés sont approuvés en clinique. Cependant l'efficacité de ces anticorps contre des tumeurs solides reste limitée et les traitements sont souvent combinés avec de la chimiothérapie. L'immunothérapie spécifique peut également être cellulaire et exploiter par immunisation active le développement de lymphocytes T cytotoxiques (CTL) capables de détruire spécifiquement les cellules malignes. De telles «vaccinations »sont actuellement testées en clinique, mais jusqu'à présent elles n'ont pas abouti aux résultats satisfaisants. Pour obtenir une réponse lymphocytaire T cytotoxique antitumorale, la cellule T doit reconnaître un antigène associé à la tumeur, présenté sous forme de peptide dans un complexe majeur d'histocompatibilité de classe I (CHM I). Cependant les cellules tumorales sont peu efficace dans la présentation d'antigène, car souvent elles se caractérisent par une diminution ou une absence d'expression des molécules d'histocompatibilité de classe I, et expriment peu ou pas de molécules d'adhésion et de cytokines costimulatrices. C'est en partie pourquoi, malgré l'induction de fortes réponses CTL spécifiquement dirigés contre des antigènes tumoraux, les régressions tumorales obtenus grâce à ces vaccinations sont relativement rares. Les lymphocytes «invariant Natural Killer T » (iNKT) forment une sous-population particulière de lymphocytes T reconnaissant des antigènes glycolipidiques présentés sur la molécule non-polymorphique CD1d, analogue aux protéines CMH I. Après activation avec le ligand de haute affinité α-galactosylceramide (αGalCer), les cellules iNKT produisent des grandes quantités de la cytokine pro-inflammatoire interferon gamma (IFN-γ) et activent les cellules du système immunitaire inné et acquis, telles que les cellules dendritiques (DC), NK et T. En conséquence, on a montré que les cellules iNKT exercent des activités anti-tumorales et la recherche s'est intéressée à la manipulation de ces cellules pour développer des thérapies anti-tumorales. Néanmoins, le désavantage majeur de l'αGalCer, injecté seul, est qu'une seule dose de ce ligand aboutit à une activation des cellules iNKT de courte durée suivie par un état anergique prolongé, limitant l'utilisation thérapeutique de ce glycolipide. Notre groupe de recherche a donc eu l'idée de développer une nouvelle approche thérapeutique où la réponse immunitaire des cellules iNKT serait prolongée et redirigée vers la tumeur par des anticorps monoclonaux. Concrètement, nous avons produit des molécules récombinantes soluble CD1d (sCD1d) qui, si elles sont chargés avec l'αGalCer (αGalCer/sCDld), aboutissent à une activation prolongée des cellules iNKT et NK associée avec la sécrétion d'IFN-γ et la maturation des cellules DC. Plus important, si la molécule αGalCer/sCD1d est fusionnée avec un fragment single-chain (scFv) de l'anticorps anti-HER2, la réponse immunitaire est redirigée à la tumeur pour autant que les cellules cancéreuses expriment l'antigène HER2. Les molécules αGalCer/sCDld ainsi présentées activent les lymphocytes iNKT. Avec cette stratégie, on observe une importante inhibition de métastases expérimentales aux poumons et de tumeurs sous-cutanées, même lorsque le traitement systémique est commencé 2 à 7 jours après la greffe des cellules de mélanome B16 transfectées avec l'antigène HER2. Dans les mêmes conditions le traitement avec l'αGalCer seul est inefficace. L'activité anti-tumorale de la protéine sCDld-anti-HER2 est associée à son accumulation spécifique dans des tumeurs exprimant le HER2 ainsi qu'avec une accumulation des cellules iNKT, NK et T à la tumeur. En conclusion, l'activation prolongée des cellules iNKT redirigées à la tumeur par des molécules récombinantes αGalCer/sCD1d conduit à l'activation de la réponse innée et adaptative au site tumoral, offrant une nouvelle stratégie prometteuse d'immunothérapie contre le cancer.

Targeting of secretory IgA to Peyer's patch dendritic and T cells after transport by intestinal M cells.

In addition to being instrumental to the protection of mucosal epithelia, secretory IgA (SIgA) adheres to and is transported by intestinal Peyer's patch (PP) M cells. The possible functional reason for this transport is unknown. We have thus examined in mice the outcome of SIgA delivered from the intestinal lumen to the cells present in the underlying organized mucosa-associated lymphoreticular tissue. We show selective association of SIgA with dendritic cells and CD4(+) T and B lymphocytes recovered from PP in vitro. In vivo, exogenously delivered SIgA is able to enter into multiple PP lining the intestine. In PP, SIgA associates with and is internalized by dendritic cells in the subepithelial dome region, whereas the interaction with CD4(+) T cells is limited to surface binding. Interaction between cells and SIgA is mediated by the IgA moiety and occurs for polymeric and monomeric molecular forms. Thus, although immune exclusion represents the main function of SIgA, transport of the Ab by M cells might promote Ag sampling under neutralizing conditions essential to the homeostasis of mucosal surfaces.

Alveolar macrophages and lung dendritic cells sense RNA and drive mucosal IgA responses.

The mechanisms regulating systemic and mucosal IgA responses in the respiratory tract are incompletely understood. Using virus-like particles loaded with single-stranded RNA as a ligand for TLR7, we found that systemic vs mucosal IgA responses in mice were differently regulated. Systemic IgA responses following s.c. immunization were T cell independent and did not require TACI or TGFbeta, whereas mucosal IgA production was dependent on Th cells, TACI, and TGFbeta. Strikingly, both responses required TLR7 signaling, but systemic IgA depended upon TLR7 signaling directly to B cells whereas mucosal IgA required TLR7 signaling to lung dendritic cells and alveolar macrophages. Our data show that IgA switching is controlled differently according to the cell type receiving TLR signals. This knowledge should facilitate the development of IgA-inducing vaccines.

Memory and effector CD8 T-cell responses after nanoparticle vaccination of melanoma patients.

Induction of cytotoxic CD8 T-cell responses is enhanced by the exclusive presentation of antigen through dendritic cells, and by innate stimuli, such as toll-like receptor ligands. On the basis of these 2 principles, we designed a vaccine against melanoma. Specifically, we linked the melanoma-specific Melan-A/Mart-1 peptide to virus-like nanoparticles loaded with A-type CpG, a ligand for toll-like receptor 9. Melan-A/Mart-1 peptide was cross-presented, as shown in vitro with human dendritic cells and in HLA-A2 transgenic mice. A phase I/II study in stage II-IV melanoma patients showed that the vaccine was well tolerated, and that 14/22 patients generated ex vivo detectable T-cell responses, with in part multifunctional T cells capable to degranulate and produce IFN-γ, TNF-α, and IL-2. No significant influence of the route of immunization (subcutaneous versus intradermal) nor dosing regimen (weekly versus daily clusters) could be observed. It is interesting to note that, relatively large fractions of responding specific T cells exhibited a central memory phenotype, more than what is achieved by other nonlive vaccines. We conclude that vaccination with CpG loaded virus-like nanoparticles is associated with a human CD8 T-cell response with properties of a potential long-term immune protection from the disease.

Correlation of tumor-associated antigens MAGE, NY-ESO-1 and P53 expression with clinical and pathological relationships of patients with oral squamous cell carcinoma

Despite advances in the diagnosisand treatment of head and neck cancer,survival rates have not improvedover recent years. New therapeuticstrategies, including immunotherapy,are the subject of extensive research.In several types of tumors, the presenceof tumor infiltrating lymphocytes(TILs), notably CD8+ T cellsand dendritic cells, has been correlatedwith improved prognosis. Moreover,some T cells among TILs havebeen shown to kill tumor cells in vitroupon recognition of tumor-associatedantigens. Tumor associated antigensare expressed in a significant proportionof squamous cell carcinoma ofthe head and neck and apparently mayplay a role in the regulation of cancercell growth notably by inhibition ofp53 protein function in some cancers.The MAGE family CT antigens couldtherefore potentially be used as definedtargets for immunotherapy andtheir study bring new insight in tumorgrowth regulation mechanisms. Between1995 - 2005 54 patients weretreated surgically in our institution forsquamous cell carcinoma of the oralcavity. Patient and clinical data wasobtained from patient files and collectedinto a computerized database.For each patient, paraffin embeddedtumor specimens were retrieved andexpression of MAGE CT antigens,p53, NY-OESO-1 were analyzed byimmunohistochemistry. Results werethen correlated with histopathologicalparameter such as tumor depth,front invasion according to Bryne andboth, local control and disease freesurvival. MAGE-A was expressed in52% of patients. NY-ESO-1 and p53expression was found in 7% and 52%cases respectively. A higher tumordepth was significantly correlatedwith expression of MAGE-Aproteins(p = 0.03). No significant correlationcould be made between the expressionof both p53 andNY-OESO-1 andhistopathological parameters. Expressionof tumor-associated antigendid not seem to impact significantlyon patient prognosis. As does thedemonstration of p53 function inhibitionby CT antigens of MAGE family,our results suggest, that tumor associatedantigens may be implicated in tumorprogression mechanisms. Thishypothesis need further investigationto clarify the relationship betweenhost immune response and local tumorbiology.

Plasmacytoid dendritic cells: one-trick ponies or workhorses of the immune system?

Plasmacytoid dendritic cells (pDCs) were first described as interferon-producing cells and, for many years, their overlapping characteristics with both lymphocytes and classical dendritic cells (cDCs) created confusion over their exact ontogeny. In this Viewpoint article, Nature Reviews Immunology asks five leaders in the field to discuss their thoughts on the development and functions of pDCs--do these cells serve mainly as a major source of type I interferons or do they also make other important contributions to immune responses?

Selective induction of NK cell proliferation and cytotoxicity by activated NKT cells.

NK T cells produce cytokines when their semi-invariant TCR engages glycolipids associated with CD1d. The physiological consequences of NKT cell activation remain controversial, although they have been implicated in control of autoimmunity, parasites and tumors. We show here that specific activation of NKT cells in liver and spleen leads to a rapid induction of extensive NK cell proliferation and cytotoxicity. This NK cell activation is dependent, at least in part, on IFN-gamma production by NKT cells and IL-12 production by antigen-presenting cells. Remarkably, activation of NK cells by NKT cells is highly selective, since bystander T and B lymphocytes show transient expression of activation markers but almost no proliferation. Collectively our data suggest that CD1d-dependent NKT cells regulate innate immunity by sampling blood-borne glycolipid antigens and rapidly activating NK cells.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Oxidative stress and inflammation response after nanoparticle exposure : differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

Combustion-derived and manufactured nanoparticles (NPs) are known to provoke oxidative stress and inflammatory responses in human lung cells; therefore, they play an important role during the development of adverse health effects. As the lungs are composed of more than 40 different cell types, it is of particular interest to perform toxicological studies with co-cultures systems, rather than with monocultures of only one cell type, to gain a better understanding of complex cellular reactions upon exposure to toxic substances. Monocultures of A549 human epithelial lung cells, human monocyte-derived macrophages and monocyte-derived dendritic cells (MDDCs) as well as triple cell co-cultures consisting of all three cell types were exposed to combustion-derived NPs (diesel exhaust particles) and to manufactured NPs (titanium dioxide and single-walled carbon nanotubes). The penetration of particles into cells was analysed by transmission electron microscopy. The amount of intracellular reactive oxygen species (ROS), the total antioxidant capacity (TAC) and the production of tumour necrosis factor (TNF)-a and interleukin (IL)-8 were quantified. The results of the monocultures were summed with an adjustment for the number of each single cell type in the triple cell co-culture. All three particle types were found in all cell and culture types. The production of ROS was induced by all particle types in all cell cultures except in monocultures of MDDCs. The TAC and the (pro-)inflammatory reactions were not statistically significantly increased by particle exposure in any of the cell cultures. Interestingly, in the triple cell co-cultures, the TAC and IL-8 concentrations were lower and the TNF-a concentrations were higher than the expected values calculated from the monocultures. The interplay of different lung cell types seems to substantially modulate the oxidative stress and the inflammatory responses after NP exposure. [Authors]

Factors determining the spontaneous activation of splenic dendritic cells in culture.

Dendritic cells (DCs) serve as a link between the innate and adaptive immune systems. The activation state of DCs is crucial in this role. However, when DCs are isolated from lymphoid tissues, purified and placed in culture they undergo 'spontaneous' activation. The basis of this was explored, using up-regulation of DC surface MHC II, CD40, CD80 and CD86 as indicators of DC activation. No evidence was found for DC damage during isolation or for microbial products causing the activation. The culture activation of spleen DCs differed from that of Langerhans cells when released from E-cadherin-mediated adhesions, since E-cadherin was not detected and activation still occurred with β-catenin null DCs. Much of the activation could be attributed to DC-DC interactions. Although increases in surface MHC II levels occurred under all culture conditions tested, the increase in expression of CD40, CD80 and CD86 was much less under culture conditions where such interactions were minimised. DC-to-DC contact under the artificial conditions of high DC concentration in culture induced the production of soluble factors and these, in turn, induced the up-regulation of co-stimulatory molecules on the DC surface.

T cell receptor specificity is critical for the development of epidermal gammadelta T cells.

A particular feature of gammadelta T cell biology is that cells expressing T cell receptor (TCR) using specific Vgamma/Vdelta segments are localized in distinct epithelial sites, e.g., in mouse epidermis nearly all gammadelta T cells express Vgamma3/Vdelta1. These cells, referred to as dendritic epidermal T cells (DETC) originate from fetal Vgamma3+ thymocytes. The role of gammadelta TCR specificity in DETC's migration/localization to the skin has remained controversial. To address this issue we have generated transgenic (Tg) mice expressing a TCR delta chain (Vdelta6.3-Ddelta1-Ddelta2-Jdelta1-Cdelta), which can pair with Vgamma3 in fetal thymocytes but is not normally expressed by DETC. In wild-type (wt) Vdelta6.3Tg mice DETC were present and virtually all of them express Vdelta6.3. However, DETC were absent in TCR-delta(-/-) Vdelta6.3Tg mice, despite the fact that Vdelta6.3Tg gammadelta T cells were present in normal numbers in other lymphoid and nonlymphoid tissues. In wt Vdelta6.3Tg mice, a high proportion of in-frame Vdelta1 transcripts were found in DETC, suggesting that the expression of an endogenous TCR-delta (most probably Vdelta1) was required for the development of Vdelta6.3+ epidermal gammadelta T cells. Collectively our data demonstrate that TCR specificity is essential for the development of gammadelta T cells in the epidermis. Moreover, they show that the TCR-delta locus is not allelically excluded.

The use of the murine model of infection with Leishmania major to reveal the antagonistic effects that IL-4 can exert on T helper cell development and demonstrate that these opposite effects depend upon the nature of the cells targeted for IL-4 signaling.

In contrast to mice from the majority of inbred strains, BALB mice develop aberrant Th2 responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of Interleukin 4, during the first 2 d of infection, by CD4+ T cells that express the Vbeta4-Valpha8 T cell receptors specific for a dominant I-A(d) restricted epitope of the LACK antigen from L. major. In contrast to this well established role of IL-4 in Th2 cell maturation, we have recently shown that, when limited to the initial period of activation of dendritic cells by L. major preceding T cell priming, IL-4 directs DCs to produce IL-12, promotes Th1 cell maturation and resistance to L. major in otherwise susceptible BALB/c mice. Thus, the antagonistic effects that IL-4 can have on Th cell development depend upon the nature of the cells (DCs or primed T cells) targeted for IL-4 signaling.

Intestinal bacteria condition dendritic cells to promote IgA production.

Immunoglobulin (Ig) A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role in limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. We show in mice that Peyer's Patch-derived dendritic cells (PP-DC) exhibit a specialized phenotype allowing the promotion of IgA production by B2 cells. This phenotype included increased expression of the retinaldehyde dehydrogenase 1 (RALDH1), inducible nitric oxide synthase (iNOS), B cell activating factor of the tumor necrosis family (BAFF), a proliferation-inducing ligand (APRIL), and receptors for the neuropeptide vasoactive intestinal peptide (VIP). The ability of PP-DC to promote anti-CD40 dependent IgA was partially dependent on retinoic acid (RA) and transforming growth factor (TGF)-beta, whilst BAFF and APRIL signaling were not required. Signals delivered by BAFF and APRIL were crucial for CD40 independent IgA production, although the contribution of B2 cells to this pathway was minimal. The unique ability of PP-DC to instruct naïve B cells to differentiate into IgA producing plasma cells was mainly imparted by the presence of intestinal commensal bacteria, and could be mimicked by the addition of LPS to the culture. These data indicate that exposure to pathogen-associated molecular patterns present on intestinal commensal bacteria condition DC to express a unique molecular footprint that in turn allows them to promote IgA production.

Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node microenvironment.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.

From the Cover: Nanoparticle conjugation of antigen enhances cytotoxic T-cell responses in pulmonary vaccination.

The ability of vaccines to induce memory cytotoxic T-cell responses in the lung is crucial in stemming and treating pulmonary diseases caused by viruses and bacteria. However, most approaches to subunit vaccines produce primarily humoral and only to a lesser extent cellular immune responses. We developed a nanoparticle (NP)-based carrier that, upon delivery to the lung, specifically targets pulmonary dendritic cells, thus enhancing antigen uptake and transport to the draining lymph node; antigen coupling via a disulfide link promotes highly efficient cross-presentation after uptake, inducing potent protective mucosal and systemic CD8(+) T-cell immunity. Pulmonary immunization with NP-conjugated ovalbumin (NP-ova) with CpG induced a threefold enhancement of splenic antigen-specific CD8(+) T cells displaying increased CD107a expression and IFN-γ production compared with immunization with soluble (i.e., unconjugated) ova with CpG. This enhanced response was accompanied by a potent Th17 cytokine profile in CD4(+) T cells. After 50 d, NP-ova and CpG also led to substantial enhancements in memory CD8(+) T-cell effector functions. Importantly, pulmonary vaccination with NP-ova and CpG induced as much as 10-fold increased frequencies of antigen-specific effector CD8(+) T cells to the lung and completely protected mice from morbidity following influenza-ova infection. Here, we highlight recruitment to the lung of a long-lasting pool of protective effector memory cytotoxic T-cells by our disulfide-linked antigen-conjugated NP formulation. These results suggest the reduction-reversible NP system is a highly promising platform for vaccines specifically targeting intracellular pathogens infecting the lung.